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Revision as of 14:32, 7 September 2012

Digestion Protocol For Plasmid Backbone Using EcoRI and PstI

-Reaction-

- 8 uL DNA linearized plasmid Backbone (25 ng/uL)

- 1 uL PstI

- 1 uL EcoRI

- 2.5 uL Buffer 10x must be a common buffer for EcoRI and PstI (e.g. buffer H in Roche system).

- X uL mQ Water

TOTAL 25 uL

Mix by pipetting when both enzymes have been added. Avoid vortexing. Enzymes are kept in cooler or ice throughout all experiments.

-- The digestion mixture is kept for 3 hours at 37 ° C

-- The mixture is kept for 20 minutes at 80ºC

Digestion Protocol For Plasmid pUC57+ Construction Using EcoRI and PstI

-Reaction-

- 96 uL pDNA

- 4 uL mQ water

- 12 uL Buffer 10x must be a common buffer for EcoRI and PstI (e.g. buffer H in Roche system).

- 2 uL + 2 uL PstI

- 2 uL + 2 uL EcoRI

TOTAL 120 uL

The volume used is so high because after digestion we were going to purified the different inserts from an agarose gel.

• Restriction enzymes will be added one by one (first 2 uL of EcoRI, after an hour other 2 uL of EcoRI and then we did the same with PstI) each hour.

Transformation Protocol Using Heat Shock

1) Take competent E.coli cells from –80°C freezer.

2) Turn on water bath to 42°C.

3) Put competent cells in a 1.5 ml tube (Eppendorf or similar). For transforming a DNA construct, use 50 ul of competent cells. For transforming a ligation, use 100 ul of competent cells. You may need more or less cells, depending how competent they are.

4) Keep tubes on ice.

5) Add 50 ng of circular DNA into E.coli cells. Incubate on ice for 30 min. to thaw competent cells.

6) Put tube(s) with DNA and E.coli into water bath at 42°C for 90 seconds.

7) Put tubes back on ice for 2 minutes to reduce damage to the E.coli cells.

8) Add 1 ml of LB (with no antibiotic added)or SOC media. Incubate tubes for 1 hour at 37°C. (Can incubate tubes for 30 minutes, unless trying to grow DNA for ligation which is more sensitive. For ligation, leave tubes for 1 hour).

9) Spread about 100 ul of the resulting culture on LB plates (with appropriate antibiotic added. Grow overnight.

10) Pick colonies about 12-16 hours later.

Ligation Protocol

-Reaction-

- "X" uL plasmid DNA*

- "Y" uL insert*

- 1uL Ligase

- 1uL Ligase Buffer

TOTAL 10 uL

The ratio that has to exist between the number of molecules of plasmid DNA and insert is 1:3 (the volumes depends on the concentration of DNAp and the insert).

Plasmid DNA extraction and purification protocol

Mini-preps. Purification protocol.

1. Different cultures (each one with a different construction), which are growing in a selective media (LB + Ampicillin), get centrifuged at 4500g, 5 min.

2. Supernatant is removed.

3. The cells can be washed (x2) with a saline solution (PBS) in order to remove impurities.

4. The pellet is resuspended in 250 μL of Resuspension Solution (RNase A added to it previously. This solution is kept at 4ºC).

Important: resuspend it completely.

5. Transfer the suspension to an eppendorf tube.

6. Add 250 μL of Lysis Solution.

7. Mix it inverting the tube 4-6 times (DO NOT VORTEX!) until solution gets viscous and slightly clear. Important: Do not incubate more than 5 min.

8. Add 350 μL of Neutralization Solution.

9. Mix it inverting the tube 4-6 times. Incubate in ice for 15-30 min.

Now if it was necessary, the process could stop here keeping the eppendorf tube in ice.

10. Centrifuge 10’ (max. rpm) in order to pellet cell debris and chromosomal DNA.

11. Transfer the supernatant (≈ 800 μL) to the spin column (pipetting to avoid carrying impurities). Important: DO NOT TRANSFERING THE PRECIPITATE!

12. Centrifuge 1’.

13. Flow-though liquid is removed.

14. Add 500 μL of Wash Solution (Solution stock has to be perfectly closed, it contains ethanol!).

15. Centrifuge ≈ 1’.

16. Flow-though liquid is removed.

17. 14, 15, 16 steps are repeated.

18. Centrifuge 1’ in order to eliminate residual Wash Solution.

19. The spin column is transferred into an eppendorf tube (the collection tube is eliminated).

20. Add 50 μL of Elution Buffer to the center of spin column membrane and let it 5’ getting soaked (it increases the efficiency of process). Important: DO NOT CONTACT THE COLUMN MEMBRANE WITH THE PIPETTE TIP!

21. Centrifuge ≈ 2’.

22. To increase the efficiency (≈ 20%) we can get the flow-though liquid and repeat the steps previously described (20 and 21).

23. The column is discarded and the solution which contains the purified plasmid can be stored in cold.